GRF analogs II

ABSTRACT

Human pancreatic GRF(hpGRF) and rat hypothalamic GRF(rhGRF) have been earlier characterized and synthesized. The invention provides synthetic peptides which are extremely potent in stimulating the release of pituitary GH in animals, including humans, which are resistant to enzymatic degradation in the body, and which have the sequence: R 1  -Ala-R 3  -Ala-Ile-Phe-Thr-R 8  -Ser-R 10  -Arg-R 12  -R 13  -Leu-R 15  -Gln-Leu-R 18  -Ala-Arg-Lys-Leu-Leu-R 24  -R 25  -Ile-R 27  -R 28  -Arg-Gln-Gln-Gly-Glu-R 34  -Asn-Gln-Glu-R 38  -R 39  -R 40  -Arg-R 42  -R 43  -R 44  -Y wherein R 1  is Tyr, D-Tyr, Met, Phe, D-Phe, pCl-Phe, Leu, His or D-His having either a C a  Me or N a  Me substitution; R 3  is Asp or D-Asp; R 8  is Ser, Asn, D-Ser or D-Asn; R 10  is Tyr or D-Tyr; R 12  is Arg or Lys; R 13  is Ile or Val; R 15  is Gly or D-Ala; R 18  is Tyr or Ser; R 24  is His or Gln; R 25  is Glu or Asp; R 27  is Met, Ala, Nle, Ile, Leu, Nva or Val; R 28  is Asn or Ser; R 34   is Arg or Ser; R 38  is Gln or Arg; R 39  is Arg or Gly; R 40  is Ser or Ala; R 42  is Phe or Ala; R 43  is Asn or Arg; R 44  is a natural amino acid. Any or all of the residues between R 28  and R 44 , inclusive, can be deleted, and the carboxyl moiety of the amino acid residue at the C-terminus can be the radical --COOR,--CRO,--CONHNHR,--CON(R) (R&#39;) or --CH 2  OR, with R and R&#39; being lower alkyl, fluoro lower alkyl or hydrogen. These peptides as well as nontoxic salts thereof may be administered to animals, including humans and cold-blooded animals, to stimulate the release of GH and may be used diagnostically.

This invention was made with Government support under Grant No. AM26741awarded by the National Institutes of Health. The Government has certainrights in this invention.

This application is a continuation-in-part of our earlier application,Ser. No. 532,170, filed Sept. 13, 1983, now abandoned.

The present invention relates to peptides having influence on thefunction of the pituitary gland in humans and other animals. Inparticular, the present invention is directed to a peptide whichpromotes the release of growth hormone by the pituitary gland.

BACKGROUND OF THE INVENTION

Physiologists have long recognized that the hypothalamus controls allthe secretory functions of the adenohypophysis with the hypothalamusproducing special polypeptides which trigger the secretion of eachpituitary hormone. Hypothalamic releasing factors have beencharacterized for the pituitary hormones thyrotropin and prolactin (thetripeptide TRF), for the pituitary gonadotropins luteinizing hormone andfollicle stimulating hormone (the decapeptide LRF, LH-RH or GnRH) andfor the pituitary hormones β-endorphin and adrenocorticotropin (the41-amino acid polypeptide CRF). An inhibitory factor has also beencharacterized in the form of somatostatin which inhibits the secretionof growth hormone(GH). In 1982, human pancreas GH releasing factors(hpGRF) were isolated from extracts of human pancreatic tumors,purified, characterized, synthesized and tested, which were found topromote the release of GH by the pituitary. Subsequently humanhypothalamic GH releasing factor (hGRF) was characterized and found tohave the same structure as hpGRF (1-44)-NH₂. Each of thesehypophysiotropic factors has been reproduced by total synthesis, andanalogs of the native structures have been synthesized. A correspondingrat hypothalamic GH releasing factor (rhGRF) has also been characterizedand synthesized.

SUMMARY OF THE INVENTION

Synthetic polypeptides have now been synthesized and tested whichrelease GH from cultured pituitary cells and which resist enzymaticdegradation in the body. These peptides have one of the followingresidues in the 1-position: Tyr, D-Tyr, Met, Phe, D-Phe, pCl-Phe, Leu,His and D-His, which has a methyl substitution either on thealpha-carbon or in the alpha amino group. They may optionally have D-Aspat the 3-position and/or D-Arg or D-Ser at the 8-position and may haveother substitutions in the molecule which we have found to promotebiological activity.

Pharmaceutical compositions in accordance with the invention includesuch analogs which are between about 27 and 44 residues in length, or anontoxic salt of any of these, dispersed in a pharmaceutically orveterinarily acceptable liquid or solid carrier. Such pharmaceuticalcompositions can be used in clinical medicine, both human andveterinary, for administration for therapeutic purposes, and alsodiagnostically. Moreover, they can be used to promote the growth ofwarm-blooded animals, including fowl, and in aquiculture forcold-blooded animals, e.g. fish, eels, etc.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

The nomenclature used to define the peptides is that specified bySchroder & Lubke, "The Peptides", Academic Press (1965), wherein inaccordance with conventional representation the amino group at theN-terminus appears to the left and the carboxyl group at the C-terminusto the right. By natural amino acid is meant one of common, naturallyoccurring amino acids found in proteins comprising Gly, Ala, Val, Leu,Ile, Ser, Thr, Lys, Arg, Asp, Asn, Glu, Gln, Cys, Met, Phe, Tyr, Pro,Trp and His. By Nle is meant norleucine, and by Nva is meant norvaline.Where the amino acid residue has isomeric forms, it is the L-form of theamino acid that is represented unless otherwise expressly indicated. ByC^(a) Me/4Cl-Phe, for example, is meant a Phe residue the alpha carbonatom of which is substituted with methyl and the phenyl group of whichis substituted with Cl in the para-position.

The invention provides synthetic peptides having the following sequence:H-R₁ -Ala-R₃ -Ala-Ile-Phe-Thr-R₈ -Ser-R₁₀ -Arg-R₁₂ -R₁₃ -Leu-R₁₅-Gln-Leu-R₁₈ -Ala-Arg-Lys-Leu-Leu-R₂₄ -R₂₅ -Ile-R₂₇ -R₂₈-Arg-Gln-Gln-Gly-Glu-R₃₄ -Asn-Gln-Glu-R₃₈ -R₃₉ -R₄₀ -Arg-R₄₂ -R₄₃ -R₄₄wherein R₁ is Tyr, D-Tyr, Met, Phe, D-Phe, pCl-Phe, Leu, His or D-Hishaving either a C^(a) Me or N^(a) Me substitution; R₃ is Asp or D-Asp;R₈ is Ser, Asn D-Ser or D-Asn; R₁₀ is Tyr or D-Tyr; R₁₂ is Arg or Lys;R₁₃ is Ile or Val; R₁₅ is Gly or D-Ala; R₁₈ is Tyr or Ser; R₂₄ is His orGln; R₂₅ is Glu or Asp; R₂₇ is Met, Ala, Nle, Ile, Leu, Nva or Val; R₂₈is Asn or Ser; R₃₄ is Arg or Ser; R₃₈ is Gln or Arg; R₃₉ is Arg or Gly;R₄₀ is Ser or Ala; R₄₂ is Phe or Ala; R₄₃ is Asn or Arg; R₄₄ is anatural amino acid; provided however that any or all of the residuesbetween R₂₈ and R₄₄, inclusive, may be deleted. The carboxyl moiety ofthe amino acid residue at the C-terminus is preferably "Y" whichrepresents the radical --COOR,--CRO,--CONHNHR,--CON(R)(R') or --CH₂ R,with R and R' being lower alkyl, fluoro lower alkyl or hydrogen; methyl,ethyl and propyl are the preferred lower alkyl groups. Usually when aresidue is included in the 44-position, an amino acid other than Cys ischosen unless there would be a desire to form a dimer or link thesynthetic peptide to another peptide. When Met appears in position 1, itmay be preferable to have another residue in position 27.

As defined above, fragments which extend from the N-terminal throughresidue-27 have biological potency, and such biologically activefragments are considered as falling within the scope of the overallinvention. When the peptide fragment extends only to residue 27 or 28, Yshould be NH₂ or a substituted amide. When the fragment extends to oneof residues 29 thru 39, Y is preferably an amide or a substituted amidebut may be OH. When the fragment has 40 or more residues, there is noclear preference for the moiety at the C-terminus.

The peptides are synthesized by a suitable method, such as byexclusively solid-phase techniques, by partial solid-phase techniques,by fragment condensation or by classical solution couplings. Theemployment of recently developed recombinant DNA techniques may be usedto prepare a portion of an analog containing only natural amino acidresidues. For example, the techniques of exclusively solid-phasesynthesis are set forth in the textbook "Solid-Phase Peptide Synthesis",Stewart & Young, Freeman & Co., San Francisco, 1969, and are exemplifiedby the disclosure of U.S. Pat. No. 4,105,603, issued Aug. 8, 1978 toVale et al. Classical solution synthesis is described in detail in thetreatise "Methoden der Organischen Chemie (Houben-Weyl): Synthese vonPeptiden", E. Wunsch (editor) (1974) Georg Thieme Verlag, Stuttgart, W.Ger. The fragment condensation method of synthesis is exemplified inU.S. Pat. No. 3,972,859 (Aug. 3, 1976). Other available syntheses areexemplified by U.S. Pat. No. 3,842,067 (Oct. 15, 1974) and U.S. Pat. No.3,862,925 (Jan. 28, 1975). When chemical syntheses are used, it may bepreferred to synthesize peptides of 29 to 32 residues in length.

Common to such chemical syntheses is the protection of the labile sidechain groups of the various amino acid moieties with suitable protectinggroups which will prevent a chemical reaction from occurring at thatsite until the group is ultimately removed. Usually also common is theprotection of an alpha-amino group on an amino acid or a fragment whilethat entity reacts at the carboxyl group, followed by the selectiveremoval of the alpha-amino protecting group to allow subsequent reactionto take place at that location. Accordingly, it is common that, as astep in the synthesis, an intermediate compound is produced whichincludes each of the amino acid residues located in its desired sequencein the peptide chain with side-chain protecting groups linked to theappropriate residues.

Also considered to be within the scope of the present invention areintermediates of the formula: X¹ -R₁ (X or X²)-Ala-R₃(X³)-Ala-Ile-Phe-Thr(X⁴)-R₈ (X⁴ or X⁵)-Ser(X⁴)-R₁₀ (X²)-Arg(X⁶)-R₁₂ (X⁶or X⁷)-R₁₃ -Leu-R₁₅ -Gln(X⁵)-Leu-R₁₈(X²)-Ala-Arg(X⁶)-Lys(X⁷)-Leu-Leu-R₂₄ (X or X⁵)-R₂₅ (X³)-Ile-R₂₇ -R₂₈ (X⁴or X⁵)-Arg(X⁶)-Gln(X⁵)-Gln(X⁵)-Gly-Glu(X³)-R₃₄ (X⁴ orX⁶)-Asn(X⁵)-Gln(X⁵)-Glu(X³ )-R₃₈ (X⁵ or X⁶ -R₃₉ (X⁶)-R₄₀(X⁴)-Arg(X⁶)-R₄₂ -R₄₃ (X⁵ or X⁶)-R₄₄ (X⁸)-X⁹ wherein: X¹ is eitherhydrogen or an a-amino protecting group. The a-amino protecting groupscontemplated by X are those well known to be useful in the art ofstep-wise synthesis of polypeptides. Among the classes of a-aminoprotecting groups which may be employed as X¹ are (1) aromaticurethan-type protecting groups, such as fluorenylmethyloxycarbonyl(FMOC), benzyloxycarbonyl(Z) and substituted Z, such asp-chlorobenzyloxycarbonyl, p-nitrobenzyloxycarbonyl,p-bromobenzyloxycarbonyl, and p-methoxybenzyloxycarbonyl; (2) aliphaticurethan protecting groups, such as t-butyloxycarbonyl (BOC),diisopropylmethyloxycarbonyl, isopropyloxycarbonyl, ethoxycarbonyl,allyloxycarbonyl; and (3) cycloalkyl urethan-type protecting groups,such as cyclopentyloxycarbonyl, adamantyloxycarbonyl,andcyclohexyloxycarbonyl. The preferred a-amino protecting group is BOC,even when an N.sup.α Me-substituted residue is employed in the1-position.

X is hydrogen or a protecting group for the imidazole nitrogen of His,such as Tos.

X² may be a suitable protecting group for the phenolic hydroxyl group ofTyr, such as tetrahydropyranyl, tert-butyl, trityl, Bzl, CBZ, 4Br-CBZand 2,6-dichlorobenzyl(DCB). The preferred protecting group is2,6-dichlorobenzyl. X² can be hydrogen which means that there is noside-chain protecting group on the amino acid residue in that position.

X³ is hydrogen or a suitable ester-forming protecting group for thecarboxyl group of Asp or Glu, such as benzyl(OBzl), 2,6-dichlorobenzyl,methyl and ethyl.

X⁴ may be a suitable protecting group for the hydroxyl group of Thr orSer, such as acetyl, benzoyl, tert-butyl, trityl, tetrahydropyranyl,Bzl, 2,6-dichlorobenzyl and CBZ. The preferred protecting group is Bzl.X⁴ can be hydrogen, which means there is no protecting group on thehydroxyl group.

X⁵ is hydrogen or a suitable protecting group for the side chain amidogroup of Asn or Gln. It is preferably xanthyl(Xan).

X⁶ is a suitable protecting group for the guanidino group of Arg, suchas nitro, Tos, CBZ, adamantyloxycarbonyl, and BOC, or is hydrogen.

X⁷ is hydrogen or a suitable protecting group for the side chain aminogroup of Lys. Illustrative of suitable side chain amino protectinggroups are 2-chlorobenzyloxycarbonyl(2-Cl-Z), Tos, t-amyloxycarbonyl andBOC.

X⁸ is hydrogen or a suitable side-chain protecting group as generallyspecified above.

Met can optionally be protected by oxygen, but is preferably leftunprotected.

The selection of a side chain amino protecting group is not criticalexcept that generally one is chosen which is not removed duringdeprotection of the a-amino groups during the synthesis. However, forsome amino acids, e.g. His, protection is not generally necessary aftercoupling is completed, and the protecting groups may be the same.

X⁹ is a suitable protecting group for the C-terminal carboxyl group,such as the ester-forming group X³, or is an anchoring bond used insolid-phase synthesis for linking to a solid resin support, or isdes-X⁹, in which case the residue at the C-terminal has a carboxylmoiety which is Y, as defined hereinbefore. When a solid resin supportis used, it may be any of those known in the art, such as one having theformulae: --0-CH₂ -resin support, --NH-benzhydrylamine (BHA) resinsupport or --NH-paramethylbenzhydrylamine (MBHA) resin support. When theunsubstituted amide is desired, use of BHA or MBHA resin is preferred,because cleavage directly gives the amide. In case the N-methyl amide isdesired, it can be generated from an N-methyl BHA resin. Should othersubstituted amides be desired or should other groups than the free acidbe desired at the C-terminus, it may be preferable to sythesize thepeptide using classical methods as set forth in the Houben-Weyl text.

In the formula for the intermediate, at least one of the X-groups is aprotecting group or X⁹ includes resin support. Thus, the invention alsoprovides a method for manufacturing a peptide of interest by (a) forminga peptide having at least one protective group and the formula (II): X¹-R₁ (X or X²)-Ala-R₃ (X³)-Ala-Ile-Phe-Thr(X⁴)-R₈ (X⁴ or X⁵)-Ser(X⁴)-Tyr(X²)-Arg(X⁶ or X⁷)-R₁₃ -Leu-Gly-Gln(X⁵)-Leu-R₁₈(X²)-Ala-Arg(X⁶)-Lys(X⁷)-Leu-Leu-R₂₄ (X or X⁵)-R₂₅ (X³)-Ile-R₂₇ R₂₈ (X⁴or X⁵)-Arg(X⁶)-Gln(X⁵)-Gln(X⁵)-Gly-Glu(X³)-R₃₄ (X⁴ orX⁶)-Asn(X⁵)-Gln(X⁵)-Glu(X³)-R₃₈ (X⁵ or X⁶)-R₃₉ (X⁶)-R₄₀ (X⁴)-Arg(X⁶)-R₄₂-R₄₃ (X⁵ or X⁶ (-R₄₄ (X⁸)-X⁹ wherein: X, X¹, X², X³, X⁴, X⁵, X⁶, X⁷ andX⁸ are each either hydrogen or a protective group and X⁹ is either aprotective group or an anchoring bond to resin support or des-X⁹, inwhich case the residue at the C-terminal has a carboxy moiety which isY; (b) splitting off the protective group or groups or anchoring bondfrom said peptide of the formula (II); and (c) if desired, converting aresulting peptide into a nontoxic salt thereof.

In selecting a particular side chain protecting group to be used in thesynthesis of the peptides, the following general rules are followed: (a)the protecting group preferably retains its protecting properties and isnot be split off under coupling conditions, (b) the protecting groupshould be stable to the reagent and, with the exception of Xan, ispreferably stable under the reaction conditions selected for removingthe a-amino protecting group at each step of the synthesis, and (c) theside chain protecting group must be removable, upon the completion ofthe synthesis containing the desired amino acid sequence, under reactionconditions that will not undesirably alter the peptide chain.

When peptides are not prepared using recombinant DNA technology, theyare preferably prepared using solid phase synthesis, such as thatgenerally described by Merrifield, J. Am. Chem. Soc., 85, p 2149 (1963),although other equivalent chemical syntheses known in the art can alsobe used as previously mentioned. Solid-phase synthesis is commenced fromthe C-terminal end of the peptide by coupling a protected a-amino acidto a suitable resin. Such a starting material can be prepared byattaching an a-amino-protected amino acid by an ester linkage to achloromethylated resin or a hydroxymethyl resin, or by an amide bond toa BHA resin or MBHA resin. The preparation of the hydroxymethyl resin isdescribed by Bodansky et al., Chem. Ind. (London) 38, 1597-98 (1966).Chloromethylated resins are commercially available from Bio RadLaboratories, Richmond, California and from Lab. Systems, Inc. Thepreparation of such a resin is described by Stewart et al., "Solid PhasePeptide Synthesis" (Freeman & Co., San Francisco 1969), Chapter 1, pp1-6. BHA and MBHA resin supports are commercially available and aregenerally used only when the desired polypeptide being synthesized hasan unsubstituted amide at the C-terminal.

The C-terminal amino acid, i.e. Asn, protected by BOC and by Xan, can befirst coupled to the chloromethylated resin according to the procedureset forth in Chemistry Letters, K. Horiki et al. 165-168 (1978), usingKF in DMF at about 60° C. for 24 hours with stirring, when the43-residue peptide is to be synthesized. Following the coupling of theBOC-protected amino acid to the resin support, the a-amino protectinggroup is removed, as by using trifluoroacetic acid(TFA) in methylenechloride, TFA alone or HCl in dioxane. The deprotection is carried outat a temperature between about 0° C. and room temperature. Otherstandard cleaving reagents and conditions for removal of specifica-amino protecting groups may be used as described in Schroder & Lubke,"The Peptides", 1 pp 72-75 (Academic Press 1965).

After removal of the a-amino protecting group, the remaining a-amino-and side chain-protected amino acids are coupled step-wise in thedesired order to obtain the intermediate compound defined hereinbefore,or as an alternative to adding each amino acid separately in thesynthesis, some of them may be coupled to one another prior to additionto the solid phase reactor. The selection of an appropriate couplingreagent is within the skill of the art. Particularly suitable as acoupling reagent is N,N'-dicyclohexyl carbodiimide (DCCI).

The activating reagents used in the solid phase synthesis of thepeptides are well known in the peptide art. Examples of suitableactivating reagents are carbodiimides, such asN,N'-diisopropylcarbodiimide andN-ethyl-N'-(3-dimethylaminopropyl)carbodiimide. Other activatingreagents and their use in peptide coupling are described by Schroder &Lubke supra, in Chapter III and by Kapoor, J. Phar. Sci., 59, pp 1-27(1970).

Each protected amino acid or amino acid sequence is introduced into thesolid phase reactor in about a fourfold or more excess, and the couplingmay be carried out in a medium of dimethylformamide(DMF): CH₂ Cl₂ (1:1)or in DMF or CH₂ Cl₂ alone. In cases where incomplete coupling occurs,the coupling procedure is repeated before removal of the a-aminoprotecting group prior to the coupling of the next amino acid. Thesuccess of the coupling reaction at each stage of the synthesis, ifperformed manually, is preferably monitored by the ninhydrin reaction,as described by E. Kaiser et al., Anal. Biochem. 34, 595 (1970). Thecoupling reactions can be performed automatically, as on a Beckman 990automatic synthesizer, using a program such as that reported in Rivieret al. Biopolymers, 1978, 17, pp 1927-1938.

After the desired amino acid sequence has been completed, theintermediate peptide can be removed from the resin support by treatmentwith a reagent, such as liquid hydrogen fluoride, which not only cleavesthe peptide from the resin but also cleaves all remaining side chainprotecting groups X, X², X³, X⁴, X⁵, X⁶, X⁷ and X⁸ and the anchoringbond X⁹ and also the a-amino protecting group X¹ if one is used, toobtain the peptide in the form of the free acid. If Met is present inthe sequence, the BOC protecting group is preferably first removed usingtrifluoroacetic acid (TFA)/ethanedithiol prior to cleaving the peptidefrom the resin with HF to eliminate potential S-alkylation. When usinghydrogen fluoride for cleaving, anisole and methylethyl sulfide areincluded in the reaction vessel for scavenging.

The following Example sets forth a preferred method for synthesizingpeptides by the solid-phase technique. It will of course be appreciatedthat the synthesis of a correspondingly shorter peptide fragment iseffected in the same manner by merely eliminating the requisite numberof amino acids at either end of the chain; however, it is presently feltthat biologically active fragments should contain the indicated sequenceat the N-terminus.

EXAMPLE I

The synthesis of the peptide [N^(a) MeHis¹ ]-rhGRF(1-43)-OH having theformula: N^(a)MeHis-Ala-Asp-Ala-Ile-Phe-Thr-Ser-Ser-Tyr-Arg-Arg-Ile-Leu-Gly-Gln-Leu-Tyr-Ala-Arg-Lys-Leu-Leu-His-Glu-Ile-Met-Asn-Arg-Gln-Gln-Gly-Glu-Arg-Asn-Gln-Glu-Gln-Arg-Ser-Arg-Phe-Asn-OHis conducted in a stepwise manner using a Beckman 990 peptidesynthesizer on a chloromethylated resin having a substitution range ofabout 0.1 to 0.5 mmoles/g. resin. Coupling of BOC-Asn(Xan) to the resinis performed by the general procedure set forth in Chemistry Letters,supra, using KF in DMF at about 60° C. for 24 hours with stirring, andit results in the substitution of about 0.35 mmol. Asn per gram ofresin.

After deblocking and neutralization, the peptide chain is builtstep-by-step on the resin. Deblocking, neutralization and addition ofeach amino acid is performed in general accordance with the procedureset forth in detail in Rivier, J, J. Amer. Chem. Soc., 96, 2986-2992(1974). All solvents that are used are carefully degassed by spargingwith an inert gas, e.g. helium or nitrogen, to insure the absence ofoxygen that might undesirably oxidize the sulfur of the Met residue.

Deblocking is preferably carried out in accordance with Schedule A whichfollows:

    ______________________________________                                        SCHEDULE A                                                                    Reagent            Mixing time (Min.)                                         ______________________________________                                        1. 60% TFA/2% ethanedithiol                                                                      10                                                         2. 60% TFA/2% ethanedithiol                                                                      15                                                         3. IPA/1% ethanedithiol                                                                          0.5                                                        4. Et.sub.3 N (10%) in CH.sub.2 Cl.sub.2                                                         0.5                                                        5. MeOH            0.5                                                        6. Et.sub.3 N (10%) in CH.sub.2 Cl.sub.2                                                         0.5                                                        7. MeOH (twice)    0.5                                                        8. CH.sub.2 Cl.sub.2 (twice)                                                                     0.5                                                        ______________________________________                                    

The couplings are preferably carried out as set out in Schedule B whichfollows:

    ______________________________________                                        SCHEDULE B                                                                    Reagent           Mixing time (Min.)                                          ______________________________________                                         9. DCCI          --                                                          10. Boc-amino acid                                                                              50-90                                                       11. MeOH (twice)  0.5                                                         12. CH.sub.2 Cl.sub.2 (twice)                                                                   0.5                                                         13. Ac.sub.2 O (3M) in CH.sub.2 Cl.sub.2                                                        15.0                                                        14. CH.sub.2 Cl.sub.2                                                                           0.5                                                         15. MeOH          0.5                                                         16. CH.sub.2 Cl.sub.2 (twice)                                                                   0.5                                                         ______________________________________                                    

Briefly, one to two mmol. of BOC-protected amino acid in methylenechloride is used per gram of resin, plus one equivalent of 1.0 molarDCCI in methylene chloride for two hours. When BOC-Arg(TOS) is beingcoupled, a mixture of 50% DMF and methylene chloride is used. Bzl etheris used as the hydroxyl side-chain protecting group for Ser and Thr. Theamino group of Asn or Gln is protected by Xan when DCC coupling is usedas is preferred. P-nitrophenyl ester(ONp) may also be used to activatethe carboxyl end of Asn or Gln, and for example, BOC-Asn(ONp) can becoupled overnight using one equivalent of HOBt in a 50% mixture of DMFand methylene chloride, in which case no DCC is added.2-chloro-benzyloxycarbonyl(2Cl-Z) is used as the protecting group forthe Lys side chain. Tos is used to protect the guanidino group of Argand the imidazole nitrogen of His, and the Glu or Asp side-chaincarboxyl group is protected with OBzl. The phenolic hydroxyl group ofTyr is protected with 2,6-dichlorobenzyl(DCB). At the end of thesynthesis, the following composition is obtained: BOC-NMeHis(X)-Ala-Asp(X³)-Ala-Ile-Phe-Thr(X⁴)-Ser(X⁴)-Ser(X.sup.4)-Tyr(X²)-Arg(X⁶)-Arg(X⁶)-Ile-Leu-Gly-Gln(X⁵)-Leu-Tyr(X²)-Ala-Arg(X⁶)-Lys(X⁷)-Leu-Leu-His(X)-Glu(X³)-Ile-Met-Asn(X⁵)-Arg(X⁶)-Gln(X⁵)-Gln(X⁵)-Gly-Glu(X³)-Arg(X.sup.6)-Asn(X⁵)-Gln(X⁵)-Glu(X³)-Gln(X⁵)-Arg(X⁶)-Ser(X⁴)-Arg(X⁶)-Phe-Asn(X⁵)-X⁹ wherein X is Tos, X² is DCB, X³ is OBzl, X⁴ isBzl, X⁵ is Xan, X⁶ is Tos, X⁷ is 2Cl-Z and X⁹ is --O-CH -resin support.Xan may have been partially or totally removed by TFA treatment used todeblock the a-amino protecting group.

In order to cleave and deprotect the protected peptide-resin, it istreated with 1.5 ml. anisole, 0.5 ml. methylethylsulfide and 15 ml.hydrogen fluoride(HF) per gram of peptide-resin, at -20° C. for one-halfhour and at 0° C. for one-half hour. After elimination of the HF underhigh vacuum, the resin-peptide remainder is washed alternately with drydiethyl ether and chloroform, and the peptide is then extracted withdegassed 2N aqueous acetic acid and separated from the resin byfiltration.

The cleaved and deprotected peptide is then dissolved in 0-5% aceticacid and subjected to purification which may include Sephadex G-50 finegel filtration.

The peptide is then further purified by preparative or semi-preparativeHPLC as described in Rivier et al., Peptides: Structure and BiologicalFunction, (1979) pp 125-8 and Marki et al. J. Am. Chem. Soc. 103, 3178(1981). Cartridges fitting Waters Associates prep LC-500 are packed with15-20u C₁₈ Silica from Vydac (300A). A gradient of CH₃ CN in TEAP isgenerated by a low pressure Eldex gradient maker, as described inRivier, J., J. Liq. Chromatography 1, 343-367 (1978). Thechromatographic fractions are carefully monitored by HPLC, and only thefractions showing substantial purity are pooled. Desalting of thepurified fractions, independently checked for purity, is achieved usinga gradient of CH₃ CN in 0.1% TFA. The center cut is then lyophilized toyield the desired peptide, the purity of which can be greater than 98%.

The synthesis is repeated using an MBHA resin to produce the samepeptide having an amidated C-terminus using an initial procedure asgenerally described in Vale et al. U.S. Pat. No. 4,292,313 to link Asnto the MBHA resin.

EXAMPLE II

The synthesis of a 40-residue amidated peptide having the formula:H-C^(a)MeHis-Ala-Asp-Ala-Ile-Phe-Thr-Ser-Ser-Tyr-Arg-Arg-Ile-Leu-Gly-Gln-Leu-Tyr-Ala-Arg-Lys-Leu-Leu-His-Glu-Ile-Met-Asn-Arg-Gln-Gln-Gly-Glu-Arg-Asn-Gln-Glu-Gln-Arg-Ser-NH₂is conducted in a stepwise manner using a Beckman 990 PeptideSynthesizer on an MBHA resin as generally described in Vale et al. U.S.Pat. No. 4,292,313. The peptide is judged to be substantially pure usingTLC and HPLC.

EXAMPLE III

The synthesis of a rhGRF analog, i.e. [N^(a) Me-Met¹, Leu²⁷]-rhGRF(1-43)-OH, having the formula: N^(a)MeMet-Ala-Asp-Ala-Ile-Phe-Thr-Ser-Ser-Tyr-Arg-Arg-Ile-Leu-Gly-Gln-Leu-Tyr-Ala-Arg-Lys-Leu-Leu-His-Glu-Ile-Leu-Asn-Arg-Gln-Gln-Gly-Glu-Arg-Asn-Gln-Glu-Gln-Arg-Ser-Arg-Phe-Asn-OHis conducted in a stepwise manner using a Beckman 990 PeptideSynthesizer on a chloromethylated resin, in the manner generallydescribed in Example I. The peptide is judged to be substantially pureusing TLC and HPLC.

EXAMPLE IV

The synthesis of a hpGRF analog fragment, i.e. [C^(a) MePhe(4Cl)¹]-hpGRF(1-32)-NH₂ having the formula: H-C^(a)MePhe(4Cl)-Ala-Asp-Ala-Ile-Phe-Thr-Asn-Ser-Tyr-Arg-Lys-Val-Leu-Gly-Gln-Leu-Ser-Ala-Arg-Lys-Leu-Leu-Gln-Asp-Ile-Met-Ser-Arg-Gln-Gln-Gly-NH₂is conducted in a stepwise manner using a Beckman 990 PeptideSynthesizer on an MBHA resin as in Example II. This analog is judged tobe substantially pure using TLC and HPLC.

The synthesis is repeated twice to produce [N^(a) MePhe(4Cl)¹]-hpGRF(1-32)-NH₂ and [N^(a) MePhe¹ ]-hpGRF(1-32)-NH₂.

EXAMPLE V

The synthesis of a hpGRF analog fragment i.e. [N^(a) MeTyr¹]-hpGRF(1-29)-NH₂ having the formula: N^(a)MeTyr-Ala-Asp-Ala-Ile-Phe-Thr-Asn-Ser-Tyr-Arg-Lys-Val-Leu-Gly-Gln-Leu-Ser-Ala-Arg-Lys-Leu-Leu-Gln-Asp-Ile-Met-Ser-Arg-NH₂is conducted in a stepwise manner using a Beckman 990 PeptideSynthesizer on an MBHA resin as in Example II. The peptide is judged tobe substantially pure using TLC and HPLC.

EXAMPLE VI

The synthesis of a rhGRF fragment i.e. [N^(a) MePhe¹ ]-rhGRF(1-29)-NH₂,having the formula: N^(a)MePhe-Ala-Asp-Ala-Ile-Phe-Thr-Ser-Ser-Tyr-Arg-Arg-Ile-Leu-Gly-Gln-Leu-Tyr-Ala-Arg-Lys-Leu-Leu-His-Glu-Ile-Met-Asn-Arg-NH₂is conducted in a stepwise manner using a Beckman 990 PeptideSynthesizer on an MBHA resin as in Example II. The peptide is judged tobe substantially pure using TLC and HPLC.

EXAMPLE VII

The synthesis of [N^(a) MeLeu¹,Ile²⁷ ]-hpGRF(1-32)-NH₂ having theformula: N^(a)MeLeu-Ala-Asp-Ala-Ile-Phe-Thr-Asn-Ser-Tyr-Arg-Lys-Val-Leu-Gly-Gln-Leu-Ser-Ala-Arg-Lys-Leu-Leu-Gln-Asp-Ile-Ile-Ser-Arg-Gln-Gln-Gly-NH₂is conducted in a stepwise manner using a Beckman 990 PeptideSynthesizer on an MBHA resin as in Example II. The peptide is judged tobe substantially pure using TLC and HPLC.

EXAMPLE VIII

The synthesis of a rhGRF analog fragment i.e. [C^(a) Me/4Cl-Phe¹]-rhGRF(1-29)-NH₂ having the formula: H-C^(a)MePhe(4Cl)-Ala-Asp-Ala-Ile-Phe-Thr-Ser-Ser-Tyr-Arg-Arg-Ile-Leu-Gly-Gln-Leu-Tyr-Ala-Arg-Lys-Leu-Leu-His-Glu-Ile-Met-Asn-Arg-NH₂is conducted in a stepwise manner using a Beckman 990 PeptideSynthesizer on an MBHA resin as in Example II. The peptide is judged tobe substantially pure using TLC and HPLC.

EXAMPLE IX

The synthesis of a rhGRF analog fragment i.e. [N^(a) MeTyr¹]-rhGRF(1-29)-NH₂ having the formula: H-N^(a)MeTyr-Ala-Asp-Ala-Ile-Phe-Thr-Ser-Ser-Tyr-Arg-Arg-Ile-Leu-Gly-Gln-Leu-Tyr-Ala-Arg-Lys-Leu-Leu-His-Glu-Ile-Met-Asn-Arg-NH₂is conducted in a stepwise manner using a Beckman 990 PeptideSynthesizer on an MBHA resin as in Example II. The peptide is judged tobe substantially pure using TLC and HPLC.

EXAMPLE X

The synthesis of [C^(a) MeLeu¹ ]-rhGRF-[Val⁴⁴ -NH₂ ]having the formula:H-C^(a)MeLeu-Ala-Asp-Ala-Ile-Phe-Thr-Ser-Ser-Tyr-Arg-Arg-Ile-Leu-Gly-Gln-Leu-Tyr-Ala-Arg-Lys-Leu-Leu-His-Glu-Ile-Met-Asn-Arg-Gln-Gln-Gly-Glu-Arg-Asn-Gln-Glu-Gln-Arg-Ser-Arg-Phe-Asn-Val-NH₂is conducted in a stepwise manner using a Beckman 990 PeptideSynthesizer on an MBHA resin as in Example II. The peptide is judged tobe substantially pure using TLC and HPLC.

EXAMPLE XI

The synthesis of a rhGRF analog fragment i.e. [N^(a) MeTyr¹,Nle²⁷]-rhGRF(1-29)-NH₂ having the formula: H-N^(a)MeTyr-Ala-Asp-Ala-Ile-Phe-Thr-Ser-Ser-Tyr-Arg-Arg-Ile-Leu-Gly-Gln-Leu-Tyr-Ala-Arg-Lys-Leu-Leu-His-Glu-Ile-Nle-Asn-Arg-NH₂is conducted in a stepwise manner using a Beckman 990 PeptideSynthesizer on an MBHA resin as in Example II. The peptide is judged tobe substantially pure using TLC and HPLC.

The synthesis is then repeated but instead of using an MBHA resin, anN-alkylamine resin is employed, also termed an N-ethylaminomethyl resin(NEAM resin), as described in copending U.S. patent application Ser. No.545,077 filed Oct. 24, 1983 the name of W. D. Kornreich et al., thedisclosure of which is incorporated herein by reference. About 10 gramsof the chloromethylated resin of Example I are reacted with 100 ml. ofethylamine at about 4° C. for about 24 hours with continuous stirring tochange the a-chlorobenzyl groups to N-ethyl a-aminobenzyl groups, uponwhich the peptide is then built via an initial, substituted-amidelinkage.

Upon completion of the desired peptide sequence, deprotection andcleavage from the resin is effected by treatment with HF, with anisoleas a scavenger, stirring first at 0° C. and then allowing the stirredmixture to slowly warm to room temperature over about 3 hours, aprocedure which cleaves the peptide from the resin as the ethylamide,i.e. [N^(a) MeTyr¹,Nle²⁷ ]-rhGRF(1-29)-NHCH₂ CH₃.

EXAMPLE XII

The synthesis of a hpGRF analog fragment, i.e. [N^(a) MeTyr¹]-hpGRF(1-32)-NH₂ having the formula: H-N^(a)MeTyr-Ala-Asp-Ala-Ile-Phe-Thr-Asn-Ser-Tyr-Arg-Lys-Val-Leu-Gly-Gln-Leu-Ser-Ala-Arg-Lys-Leu-Leu-Gln-Asp-Ile-Met-Ser-Arg-Gln-Gln-Gly-NH₂is conducted in a stepwise manner using a Beckman 990 PeptideSynthesizer on an MBHA resin as in Example II. This analog is judged tobe substantially pure using TLC and HPLC.

The synthesis is repeated to produce [C^(a) MeTyr¹ ]-hpGRF(1-32)-NH₂.

EXAMPLE XIII

The synthesis of a hpGRF analog fragment i.e. [N^(a) MeTyr¹,Nle²⁷, Asn²⁸]-hpGRF(1-29)-NH₂ having the formula: N^(a)MeTyr-Ala-Asp-Ala-Ile-Phe-Thr-Asn-Ser-Tyr-Arg-Lys-Val-Leu-Gly-Gln-Leu-Ser-Ala-Arg-Lys-Leu-Leu-Gln-Asp-Ile-Nle-Asn-Arg-NH₂is conducted in a stepwise manner using a Beckman 990 PeptideSynthesizer on an MBHA resin as in Example II. The peptide is judged tobe substantially pure using TLC and HPLC.

The synthesis is then repeated using an NEAM resin as set forth inExample XI. It is cleaved by treatment with HF and anisole to yield theethylamide, i.e. [N^(a) MeTyr¹,Nle²⁷, Asn²⁸ ]-hpGRF(1-29)-NHCH₂ CH₃.

EXAMPLE XIV

The synthesis of a hpGRF analog fragment i.e. [N^(a) MeTyr¹,Nle²⁷]-hpGRF(1-29)-NH₂ having the formula: N^(a)MeTyr-Ala-Asp-Ala-Ile-Phe-Thr-Asn-Ser-Tyr-Arg-Lys-Val-Leu-Gly-Gln-Leu-Ser-Ala-Arg-Lys-Leu-Leu-Gln-Asp-Ile-Nle-Ser-Arg-NH₂is conducted in a stepwise manner using a Beckman 990 PeptideSynthesizer on an MBHA resin as in Example II. The peptide is judged tobe substantially pure using TLC and HPLC.

The synthesis is then repeated using an NEAM resin as generally setforth in Example XI. It is cleaved by treatment with HF and anisole toyield the ethylamide, i.e. [N^(a) MeTyr¹,Nle²⁷ ]-hpGRF(1-29)-NHCH₂ CH₃.

EXAMPLE XV

The synthesis of a hpGRF analog fragment i.e. [N^(a) MeD-Tyr¹,Nle²⁷,Asn²⁸ ]-hpGRF(1-29)-NH₂ having the formula: N^(a)MeD-Tyr-Ala-Asp-Ala-Ile-Phe-Thr-Asn-Ser-Tyr-Arg-Lys-Val-Leu-Gly-Gln-Leu-Ser-Ala-Arg-Lys-Leu-Leu-Gln-Asp-Ile-Nle-Asn-Arg-NH₂is conducted in a stepwise manner using a Beckman 990 PeptideSynthesizer on an MBHA resin as in Example II. The peptide is judged tobe substantially pure using TLC and HPLC.

The synthesis is then repeated using an NEAM resin as generally setforth in Example XI. It is cleaved by treatment with HF and anisole toyield the ethylamide, i.e. [N^(a) MeD-Tyr¹,Nle²⁷, Asn²⁸]-hpGRF(1-29)-NHCH₂ CH₃.

EXAMPLE XVI

The synthesis of a hpGRF analog fragment i.e. [N^(a) MeD-Tyr¹,Nle²⁷]-hpGRF(1-29)-NH₂ having the formula: N^(a)MeD-Tyr-Ala-Asp-Ala-Ile-Phe-Thr-Asn-Ser-Tyr-Arg-Lys-Val-Leu-Gly-Gln-Leu-Ser-Ala-Arg-Lys-Leu-Leu-Gln-Asp-Ile-Nle-Ser-Arg-NH₂is conducted in a stepwise manner using a Beckman 990 PeptideSynthesizer on an MBHA resin as in Example II. The peptide is judged tobe substantially pure using TLC and HPLC.

The synthesis is then repeated using an NEAM resin as generally setforth in Example XI. It is cleaved by treatment with HF and anisole toyield the ethylamide, i.e. [N^(a) MeD-Tyr¹,Nle²⁷ ]-hpGRF(1-29)-NHCH₂CH₃.

EXAMPLE XVII

The synthesis of a hpGRF analog fragment i.e. [N^(a) MeTyr¹,D-Asp³,Nle²⁷, Asn²⁸ ]-hpGRF(1-29)-NH₂ having the formula: N^(a)MeTyr-Ala-D-Asp-Ala-Ile-Phe-Thr-Asn-Ser-Tyr-Arg-Lys-Val-Leu-Gly-Gln-Leu-Ser-Ala-Arg-Lys-Leu-Leu-Gln-Asp-Ile-Nle-Asn-Arg-NH₂is conducted in a stepwise manner using a Beckman 990 PeptideSynthesizer on an MBHA resin as in Example II. The peptide is judged tobe substantially pure using TLC and HPLC.

The synthesis is then repeated using an NEAM resin as generally setforth in Example XI. It is cleaved by treatment with HF and anisole toyield the ethylamide, i.e. [N^(a) MeTyrHu 1,D-Asp³, Asn²⁸]-hpGRF(1-29)-NHCH₂ CH₃.

EXAMPLE XVIII

The synthesis of a rhGRF analog fragment i.e. [N^(a) MeTyr¹,Nle²⁷, Ser²⁸]-rhGRF(1-29)-NH₂ having the formula: H-N^(a)MeTyr-Ala-Asp-Ala-Ile-Phe-Thr-Ser-Ser-Tyr-Arg-Arg-Ile-Leu-Gly-Gln-Leu-Tyr-Ala-Arg-Lys-Leu-Leu-His-Glu-Ile-Nle-Ser-Arg-NH₂is conducted in a stepwise manner using a Beckman 990 PeptideSynthesizer on an MBHA resin as in Example II. The peptide is judged tobe substantially pure using TLC and HPLC.

The synthesis is then repeated using an NEAM resin as generally setforth in Example XI. It is cleaved by treatment with HF and anisole toyield the ethylamide, i.e. [N^(a) MeTyr¹,Nle²⁷,Ser²⁸ ]-rhGRF(1-29)-NHCH₂CH₃.

EXAMPLE XIX

The synthesis of a rhGRF analog fragment i.e. [N^(a) MeD-Tyr¹,Nle²⁷]-rhGRF(1-29)-NH₂ having the formula: H-N^(a)MeD-Tyr-Ala-Asp-Ala-Ile-Phe-Thr-Ser-Ser-Tyr-Arg-Arg-Ile-Leu-Gly-Gln-Leu-Tyr-Ala-Arg-Lys-Leu-Leu-His-Glu-Ile-Nle-Asn-Arg-NH₂is conducted in a stepwise manner using a Beckman 990 PeptideSynthesizer on an MBHA resin as in Example II. The peptide is judged tobe substantially pure using TLC and HPLC.

The synthesis is then repeated using an NEAM resin as generally setforth in Example XI. It is cleaved by treatment with HF and anisole toyield the ethylamide, i.e. [N^(a) MeD-Tyr¹,Nle²⁷ ]-rhGRF(1-29)-NHCH₂CH₃.

EXAMPLE XX

The synthesis of a rhGRF analog fragment i.e. [N^(a) MeD-Tyr¹,Nle²⁷,Ser²⁸ ]-rhGRF(1-29)-NH₂ having the formula: H-N^(a)MeD-Tyr-Ala-Asp-Ala-Ile-Phe-Thr-Ser-Ser-Tyr-Arg-Arg-Ile-Leu-Gly-Gln-Leu-Tyr-Ala-Arg-Lys-Leu-Leu-His-Glu-Ile-Nle-Ser-Arg-NH₂is conducted in a stepwise manner using a Beckman 990 PeptideSynthesizer on an MBHA resin as in Example II. The peptide is judged tobe substantially pure using TLC and HPLC.

The synthesis is then repeated using an NEAM resin as generally setforth in Example XI. It is cleaved by treatment with HF and anisole toyield the ethylamide, i.e. [N^(a) MeD-Tyr¹,Nle²⁷, Ser²⁸]-rhGRF(1-29)-NHCH₂ CH₃.

EXAMPLE XXI

The synthesis of a rhGRF analog fragment i.e. [N^(a) MeTyr¹,D-Asp³,Nle²⁷ ]-rhGRF(1-29)-NH₂ having the formula: H-N^(a)MeTyr-Ala-D-Asp-Ala-Ile-Phe-Thr-Ser-Ser-Tyr-Arg-Arg-Ile-Leu-Gly-Gln-Leu-Tyr-Ala-Arg-Lys-Leu-Leu-His-Glu-Ile-Nle-Asn-Arg-NH₂is conducted in a stepwise manner using a Beckman 990 PeptideSynthesizer on an MBHA resin as in Example II. The peptide is judged tobe substantially pure using TLC and HPLC.

The synthesis is then repeated using an NEAM resin as generally setforth in Example XI. It is cleaved by treatment with HF and anisole toyield the ethylamide, i.e. [N^(a) MeTyr¹,D-Asp³, Nle²⁷]-rhGRF(1-29)-NHCH₂ CH₃.

Various of the synthetic peptides prepared in the Examples are comparedwith synthetic hpGRF(1-40)--OH in in vitro assays and are found toexhibit generally greater potencies for the secretion of GH and similarintrinsic activities.

To determine the effectiveness of the various synthetic peptides topromote the release of growth hormone, in vitro assays are carried outusing synthetic hpGRF(1-40)--OH as a standard in side-by-side comparisonwith equimolar concentrations of the various other analogs and fragmentssynthesized. Cultures are used which include cells of rat pituitaryglands removed some three to five days previously. Cultures which areconsidered optimal for the secretion of growth hormone are used for thecomparative testing, in the general manner described in Vale et al.Endocrinology , 91, 562-572 (1972) and as more particularly described inVale et al. Endocrinology, (1983), in press. Incubation with thesubstance to be tested is carried out for 3 to 4 hours, and aliquots ofthe culture medium are removed and processed to measure their contentsin immunoreactive GH(ir GH) by a well-characterized radioimmunoassay.

The results of this comparative testing for equimolar concentrations areshown in Table I.

                  TABLE I                                                         ______________________________________                                        Peptide                Comparison %                                           ______________________________________                                        hpGRF(1-40)-OH         100%                                                   (standard for this test)                                                      [N.sup.a MePhe.sup.1 ]-hpGRF(1-32)-NH.sub.2                                                           12%                                                   [C.sup.a Me/4Cl-Phe.sup.1 ]-hpGRF(1-32)-NH.sub.2                                                      5%                                                    [N.sup.a MeTyr.sup.1 ]hpGRF(1-32)-NH.sub.2                                                           184%                                                   [N.sup.a MeTyr.sup.1, Nle.sup.27 ]-rhGRF(1-29)-NH.sub.2                                              1130%                                                  ______________________________________                                    

In vitro testing of these synthetic peptides shows that they each havethe full intrinsic biological activity of hpGRF(1-40)-OH. The maximumeffective concentration for [N^(a) MePhe¹ ]-hpGRF(1-32)-NH₂ is about5-10 nanomolar.

In addition to the in vitro tests for secretion of growth hormone, invivo experiments inject the synthetic peptides through an indwellingcatheter into freely running normal male rats after pretreating themwith FLA-63, a dopamine hydroxylase inhibitor that suppressesspontaneous GH secretion without affecting the response to exogenousGRF. Blood samples are taken through the same catheter immediately priorto and 5 and 20 minutes after injections. GH levels in blood, measuredby radioimmunoassay, show that synthetic [N^(a) MePhe¹ ]-hpGRF(1-32)-NH₂and other N^(a) Me or C^(a) Me analogs thereof are powerful stimulatorsof the secretion of pituitary GH and have substantially longer durationthat hpGRF(1-32)-NH₂. Dosages between about 100 nanograms and about 50micrograms per Kg. of body weight are considered to be effective incausing secretion.

Such synthetic rhGRF and hpGRF analogs should be useful for humanapplications in which a physician wishes to elevate GH production.Stimulation of GH secretion by such analogs is of interest in patientswith complete or relative GH deficiency caused by underproduction ofendogenous GRF. Furthermore, it is probable that increased GH secretionand its attendant increase in growth could be obtained in humans oranimals with normal GH levels. Moreover, administration should alterbody fat content and modify other GH-dependent metabolic, immunologicand developmental processes. For example, these analogs may be useful asa means of stimulating anabolic processes in human beings undercircumstances such as following the incurring of burns. As anotherexample, these analogs may be administered to commercial warm-bloodedanimals, such as chickens, turkeys, pigs, goats, cattle and sheep, andmay be used in aquiculture for raising fish and other cold-bloodedmarine animals, e.g. sea turtles and eels, and amphibians, to accelerategrowth and increase the ratio of protein to fat gained by feedingeffective amounts of the peptides.

For administration to humans, these synthetic peptides should have apurity of at least about 93% and preferably at least 98%. Purity, forpurposes of this application, refers to the intended peptideconstituting the stated weight % of all peptides and peptide fragmentspresent. For the administration of such synthetic peptides to commercialand other animals in order to promote growth and reduce fat content, apurity as low as about 5%, or even as low as 0.01%, may be acceptable.

These synthetic peptides or the nontoxic salts thereof, combined with apharmaceutically or veterinarily acceptable carrier to form apharmaceutical composition, may be administered to animals, includinghumans, either intravenously, subcutaneously, intramuscularly,percutaneously, e.g. intranasally or even orally. The administration maybe employed by a physician to stimulate the release of GH where the hostbeing treated requires such therapeutic treatment. The required dosagewill vary with the particular condition being treated, with the severityof the condition and with the duration of desired treatment.

Such peptides are often administered in the form of nontoxic salts, suchas acid addition salts or metal complexes, e.g., with zinc, iron or thelike (which are considered as salts for purposes of this application).Illustrative of such acid addition salts are hydrochloride,hydrobromide, sulphate, phosphate, maleate, acetate, citrate, benzoate,succinate, malate, ascorbate, tartrate and the like. If the activeingredient is to be orally administered in tablet form, the tablet maycontain a binder, such as tragacanth, corn starch or gelatin; adisintegrating agent, such as alginic acid; and a lubricant, such asmagnesium stearate. If administration in liquid form is desired,sweetening and/or flavoring may be used, and intravenous administrationin isotonic saline, phosphate buffer solutions or the like may beeffected.

The peptides should be administered to humans under the guidance of aphysician, and pharmaceutical compositions will usually contain thepeptide in conjunction with a conventional, solid or liquid,pharmaceutically-acceptable carrier. Usually, the parenteral dosage willbe from about 100 nanograms to about 50 micrograms of the peptide perkilogram of the body weight of the host.

Although the invention has been described with regard to its preferredembodiments, which constitute the best mode presently known to theinventors, it should be understood that various changes andmodifications as would be obvious to one having the ordinary skill inthis art may be made without departing from the scope of the inventionwhich is set forth in the claims appended hereto. For example,modifications in the peptide chain, particularly deletions beginning atthe carboxyl terminal of the peptide, can be made in accordance with theknown experimental practises to date to create peptides or peptidefragments that retain all or very substantial portions of the biologicalpotency of the peptide, and such peptides are considered as being withinthe scope of the invention. Moreover, it may be possible to makeadditions to either terminus, or to both termini, and/or generallyequivalent residues can be substituted for naturally occurring residuesas is well-known in the overall art of peptide chemistry to produceother analogs having at least a substantial portion of the potency ofthe claimed polypeptide without deviating from the scope of theinvention. Preferably, there are no additions made to the N-terminusother than an C^(a) - or N^(a) -methylation. As mentioned hereinbefore,the alpha-carboxyl group of the residue at the C-terminus is preferablyamidated; however a number of equivalent moieties can also be presenthere without destroying biological potency.

Various features of the invention are emphasized in the claims whichfollow.

What is claimed is:
 1. A synthetic peptide having the formula: R₁-Ala-R₃ -Ala-Ile-Phe-Thr-R₈ -Ser-R₁₀ -Arg-R₁₂ -R₁₃ -Leu-R₁₅ -Gln-Leu-R₁₈-Ala-Arg-Lys-Leu-Leu-R₂₄ -R₂₅ -Ile-R₂₇ -R₂₈ -Arg-Gln-Gln-Gly-Glu-R₃₄-Asn-Gln-Glu-R₃₈ -R₃₉ -R₄₀ -Arg-R₄₂ -R₄₃ -R₄₄ -Y wherein R₁ is Tyr,D-Tyr, Met, Phe, D-Phe, pCl-Phe, Leu, His or D-His, which residue haseither a C^(a) Me or N^(a) Me substitution; R₃ is Asp or D-Asp; R₈ isSer, Asn D-Ser or D-Asn; R₁₀ is Tyr or D-Tyr; R₁₂ is Arg or Lys; R₁₃ isIle or Val; R₁₅ is Gly or D-Ala; R₁₈ is Tyr or Ser; R₂₄ is His or Gln;R₂₅ is Glu or Asp; R₂₇ is Met, Ala, Nle, Ile, Leu, Nva or Val; R₂₈ isAsn or Ser; R₃₄ is Arg or Ser; R₃₈ is Gln or Arg; R₃₉ is Arg or Gly; R₄₀is Ser or Ala; R₄₂ is Phe or Ala; R₄₃ is Asn or Arg; R₄₄ is Leu or Val;and Y is the carboxyl moiety of the amino acid residue at theC-terminus, which is the radical --COOR,--CRO,--CONHNHR, --CON(R)(R') or--CH₂ OR, with R and R' being lower alkyl, fluoro lower alkyl orhydrogen; or a biologically active fragment thereof extending from theN-terminus to a residue in any of positions 27 through 43 as itsC-terminus; or a nontoxic acid addition salt thereof.
 2. The peptide ofclaim 1 wherein R₁ is N^(a) MeTyr.
 3. The peptide of claim 2 wherein R₂₇is Nle.
 4. The peptide of claim 3 wherein Y is CONH₂.
 5. The peptide ofclaim 1 wherein R₃ is D-Asp.
 6. The peptide of claim 1 wherein R₁ isN^(a) MeHis.
 7. The peptide of claim 1 wherein R₁ is C^(a) MeTyr.
 8. Thepeptide of claim 1 wherein R₁ is C^(a) Me/4Cl-Phe.
 9. The peptide ofclaim 2 wherein R₃ is Asp, R₈ is Ser, R₁₀ is Tyr, R₁₂ is Arg, R₁₃ isIle, R₁₅ is Gly, R₁₈ is Tyr, R₂₄ is His, R₂₅ is Glu, R₂₇ is Met, R₂₈ isAsn, R₃₄ is Arg, R₃₈ is Gln, R₃₉ is Arg, R₄₀ is Ser, R₄₂ is Phe, R₄₃ isAsn, R₄₄ is deleted and Y is --COOH or --CONH₂.
 10. The peptide of claim2 wherein R₃ is Asp, R₈ is Ser, R₁₀ is Tyr, R₁₂ is Arg, R₁₃ is Ile, R₁₅is Gly, R₁₈ is Tyr, R₂₄ is His, R₂₅ is Glu, R₂₇ is Nle, R₂₈ is Asn, R₃₄is Arg, R₃₈ is Gln, R₃₉ is Arg, R₄₀ is Ser, R₄₂ is Phe, R₄₃ is Asn, R₄₄is deleted and Y is --COOH or --CONH₂.
 11. The peptide of claim 2wherein R₃ is Asp, R₈ is Asn, R₁₀ is Tyr, R₁₂ is Lys, R₁₃ is Val, R₁₅ isGly, R₁₈ is Ser, R₂₄ is Gln, R₂₅ is Asp, R₂₇ is Met, R₂₈ is Ser, R₃₄ isSer, R₃₈ is Arg, R₃₉ is Gly, R₄₀ is Ala, R₄₂ is Ala, R₄₃ is Arg, R₄₄ isLeu and Y is --COOH or --CONH₂.
 12. The peptide of claim 1 wherein R₁ isN^(a) Me(D-Tyr).
 13. The peptide of claim 1 having the formula of [N^(a)MeTyr¹,Nle²⁷ ]-rhGRF(1-29)-NH₂.
 14. The peptide of claim 1 having theformula [N^(a) MeTyr¹,Nle²⁷ Asn²⁸ ]-hpGRF(1-29)-NH₂.
 15. Apharmaceutical composition for stimulating the release of GH in ananimal comprising an effective amount of the peptide of claim 1 or anontoxic salt thereof, and a pharmaceutically or veterinarily acceptableliquid or solid carrier therefor.
 16. A method of stimulating therelease of growth hormone in an animal, which comprises administering tosaid animal an effective amount of a synthetic peptide having theformula: R₁ -Ala-R₃ -Ala-Ile-Phe-Thr-R₈ -Ser-R₁₀ -Arg-R₁₂ -R₁₃ -Leu-R₁₅-Gln-Leu-R₁₈ -Ala-Arg-Lys-Leu-Leu-R₂₄ -R₂₅ -Ile-R₂₇ -R₂₈-Arg-Gln-Gln-Gly-Glu-R₃₄ -Asn-Gln-Glu-R₃₈ -R₃₉ -R₄₀ -Arg-R₄₂ -R₄₃ -R₄₄-Y wherein R₁ is Tyr, D-Tyr, Met, Phe, D-Phe, pCl-Phe, Leu, His orD-His, which residue has either a C^(a) Me or N^(a) Me substitution; R₃is Asp or D-Asp; R₈ is Ser, Asn D-Ser or D-Asn; R₁₀ is Tyr or D-Tyr; R₁₂is Arg or Lys; R₁₃ is Ile or Val; R₁₅ is Gly or D-Ala; R₁₈ is Tyr orSer; R₂₄ is His or Gln; R₂₅ is Glu or Asp; R₂₇ is Met, Ala, Nle, Ile,Leu, Nva or Val; R₂₈ is Asn or Ser; R₃₄ is Arg or Ser; R₃₈ is Gln orArg; R₃₉ is Arg or Gly; R₄₀ is Ser or Ala; R₄₂ is Phe or Ala; R₄₃ is Asnor Arg; R₄₄ is Leu or Val; and Y is the carboxyl moiety of the aminoacid residue at the C-terminus, which is the radical--COOR,--CRO,--CONHNHR, --CON(R)(R') or -CH₂ OR, with R and R' beinglower alkyl, fluoro lower alkyl or hydrogen; or a biologically activefragment thereof extending from the N-terminus to a residue in any ofpositions 27 through 43 as its C-terminus; or a nontoxic acid additionsalt thereof.
 17. A method for promotion of growth in warm-bloodednonhuman animals in accordance with claim
 16. 18. A method for growthpromotion in aquiculture by administering to fish or other cold-bloodedanimals in accordance with claim
 16. 19. A method of accelerating growthin non-human animals, which method comprises administering an effectiveamount of a synthetic peptide having the sequence: R₁ -Ala-R₃-Ala-Ile-Phe-Thr-R₈ -Ser-R₁₀ -Arg-R₁₂ -R₁₃ -Leu-R₁₅ -Gln-Leu-R₁₈-Ala-Arg-Lys-Leu-Leu-R₂₄ -R₂₅ -Ile-R₂₇ -R₂₈ -R₂₉ wherein R₁ is Tyr,D-Tyr, Met, Phe, D-Phe, pCl-Phe, Leu, His or D-His which residue haseither a C^(a) Me or N^(a) Me substitution; R₃ is Asp or D-Asp; R₈ isSer, Asn, D-Ser or D-Asn; R₁₀ is Tyr or D-Tyr; R₁₂ is Arg or Lys: R₁₃ isIle or Val; R₁₅ is Gly or D-Ala; R₁₈ is Tyr or Ser; R₂₄ is His or Gln;R₂₅ is Glu or Asp; R₂₇ is Met, Ala, Nle, Ile, Leu, Nva or Val; R₂₈ isAsn, Ser or des-R₂₈ ; and R₂₉ is Arg or des-R₂₉, or a nontoxic acidaddition salt thereof.
 20. A synthetic peptide having the formula: R₁-Ala-R₃ -Ala-Ile-Phe-Thr-R₈ -Ser-R₁₀ -Arg-R₁₂ -R₁₃ -Leu-R₁₅ -Gln-Leu-R₁₈-Ala-Arg-Lys-Leu-Leu-R₂₄ -R₂₅ -Ile-R₂₇ -R₂₈ -R₂₉ -R₃₀ -R₃₁ -R₃₂ -Ywherein R₁ is Tyr, D-Tyr, Met, Phe, D-Phe, pCl-Phe, Leu, His or D-Hiswhich residue has either a C^(a) Me or N^(a) Me substitution; R₃ is Aspor D-Asp; R₈ is Ser, Asn, D-Ser or D-Asn; R₁₀ is Tyr or D-Tyr; R₁₂ isArg or Lys; R₁₃ is Ile or Val; R₁₅ is Gly or D-Ala; R₁₈ is Tyr or Ser;R₂₄ is His or Gln; R₂₅ is Glu or Asp; R₂₇ is Met, Ala, Nle, Ile, Leu,Nva or Val; R₂₈ is Asn, Ser or des-R₂₈ ; R₂₉ is Arg or des-R_(29;) R₃₀is Gln or des-R₃₀ ; R₃₁ is Gln or des-R₃₁ ; R₃₂ is Gly or des-R₃₂ ; andY is CON(R)(R') with R and R' being lower alkyl, fluoro lower alkyl orhydrogen; or a nontoxic acid addition salt thereof.